RNA Demethylase ALKBH5 Demethylates And Modμlates FUT8 Expression To Affect Keratinocyte Function

Research purposesN6-methyladenosine(m6A)is the most common and most conserved internal transcriptional modification,which is mainly coordinated by m6 A methyltransferase(writer),demethylase(eraser)and m6 A binding protein(reader).m6 A demethylation mediated by ALKBH5 regμlates gene expression by affecting mμltiple events in RNA metabolism,such as pre-mRNA processing,mRNA decay and translation.At present,most studies on ALKBH5 and disease are mainly focused on cancer,and no studies have been conducted in psoriasis.Psoriasis is a common chronic skin disease,its pathogenesis is very complex,has not been thoroughly studied,and so far there is no effective cure for psoriasis,so the study of psoriasis is still a long way to go.Research MethodsThe expression of ALKBH5 was detected in both the psoriasis case group and the healthy control group in mice and humans,and a series of cell experiments were performed in vitro to demonstrate the functional role of ALKBH5 in keratinocytes.Among them,we used CCK8(Cell Counting Kit 8)and EdU Cell proliferation to detect the effects of ALKBH5 on keratinocyte proliferation,and studied the effects of ALKBH5 on Cell migration ability and apoptosis by scrape assay and flow cytometry.Finally,we verified whether ALKBH5 acts through a demethylation mechanism.Firstly,the downstream factors are predicted by m6 A related websites,and further real-time quantitative PCR Detecting System is used.Real-time fluorescence quantitative accounting quantitative detection system)and WB(Western Blot)were used to determine whether ALKBH5 expression was regμlated by the transcriptome level and protein level,and RIP(RNA binding protein immunoprecipitation)was used to prove that ALKBH5 protein coμld bind to FUT8 mRNA.And Me RIP(methylated RNA Immunoprecipitation,methylated RNA Immunoprecipitation)was used to verify the specific mechanism of ALKBH5.Finally,the approximate location of the binding site was determined by double luciferase experiment,and the role of methylated reading protein was further explored.Research ResμltsWe found that higher ALKBH5 expression levels were observed in both the skin lesions of psoriasis model mice and in the skin lesions of clinical psoriasis patients compared with healthy controls.Secondly,in vitro cell function experiments proved that ALKBH5 knockdown improved the proliferation and migration ability of HaCaT cells and reduced their apoptosis.RIP test verified that ALKBH5 and FUT8 mRNA were definitely bound,Me RIP proved that ALKBH5’s regulation effect on FUT8 was realized through demethylation mechanism,and double luciferase test also determined the general location of binding site.Finally,it was found that methylated reading protein would recognize the m6 A site of FUT8 mRNA after knockdown ALKBH5,thus promoting its degradation.Research ConclusionsALKBH5 regμlates the expression of FUT8 through demethylation,thus playing a role in HaCaT and becoming a promoter of psoriasis.This study may provide a theoretical basis for the diagnosis and treatment of psoriasis in the future.